Cyanylation of the sulfhydryl groups of (Na ion plus K ion)-ATPase, an enzyme responsible for cation transport in cell membranes, by 5 x 10 to the minus 3rd power M 5-thiocyano-2-nitrobenzoic acid, causes approximately 30 percent inactivation. Na ion plus ATP protect the enzyme from cyanylation and inactivation, whereas phosphorylation by Na ion plus ATP plus Mg ions enhances cyanylation without affecting inactivation. The data suggest the presence of two populations of sulfhydryl groups in this ATPase. Cleavage of the major protein of the enzyme occurs at different points depending on the presence or absence of sodium during trypsin action.